Primary hyperparathyroidism (pHPT) affects up to 2% of post-menopausal women and causes decreased bone mineral density, bone fractures, and kidney stones. Despite the high prevalence and morbidity of the disease, the pathogenesis of pHPT remains unclear. Our proposed studies represent the first large-scale prospective effort to examine potentially modifiable risk factors for pHPT. We aim to produce new insights into pHPT that may lead to new approaches to prevention and treatment of this common and costly disease. The parathyroid adenomas of sporadic pHPT are monoclonal, suggesting that these neoplasms originate from single cells with a growth conferring mutation. Thus, our prospective studies of pHPT risk in Aims 1 and 2 will examine factors that chronically stimulate PTH release and/or cause parathyroid gland hyperplasia. We recently reported that lower calcium intake was associated with increased risk of incident pHPT in the Nurses Health Study (NHS) I. In Aim 1, we will conduct prospective cohort studies of > 145,000 women in NHS I and II without pHPT at baseline to delineate independent associations between body size, menopause, dietary factors (including vitamin D, magnesium, protein, and vitamin A), medications (including loop and thiazide diuretics), and the subsequent risk of incident pHPT. No study to date has prospectively examined plasma risk factors for pHPT. Using stored blood samples collected prior to diagnosis, we have the unique ability to investigate such factors in nested, prospective case- control studies in NHS I and II (N = 450 cases, 900 controls). In Aim 2, we hypothesize that higher plasma phosphorus, lower 25-hydroxyvitamin D (25[OH]D), and lower FGF23 are independently associated with increased risk of incident pHPT. We also hypothesize that unbound, or free 25(OH)D (estimated by total 25[OH]D, vitamin D binding protein, and albumin) is more strongly associated with risk than total 25(OH)D. In Aim 3, we will use tissue microarray immunohistochemical staining to quantify expression of cyclin D1 and the calcium sensing receptor (CaSR) in resected, enlarged parathyroid glands of 200 NHS I and II participants with pHPT. Cyclin D1 (cycD1), an integral component of cell-cycle regulation, is highly expressed in ~ 40% of parathyroid adenomas, and lower CaSR on enlarged parathyroid glands may represent a cause, rather than a consequence, of pHPT. We hypothesize that 1) higher cycD1 and lower CaSR expression are associated with increased parathyroid gland weight, a determinant of disease severity, 2) higher BMI, menopause, smoking, and lower use of NSAIDs are associated with higher cycD1 expression, and 3) lower cumulative intake of vitamin D and higher BMI are associated with lower CaSR expression. This will be the first study to examine associations between environmental factors assessed before pHPT diagnosis and the subsequent tissue expression of parathyroid proteins that may play important roles in the pathogenesis and/or severity of pHPT.